WO2022007326A1 - 一种用于阪崎肠杆菌o抗原实时荧光pcr检测的方法及应用 - Google Patents

一种用于阪崎肠杆菌o抗原实时荧光pcr检测的方法及应用 Download PDF

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WO2022007326A1
WO2022007326A1 PCT/CN2020/135549 CN2020135549W WO2022007326A1 WO 2022007326 A1 WO2022007326 A1 WO 2022007326A1 CN 2020135549 W CN2020135549 W CN 2020135549W WO 2022007326 A1 WO2022007326 A1 WO 2022007326A1
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nucleotide sequence
seq
enterobacter sakazakii
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郭玺
王敏
许琮
刘斌
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南开大学
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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  • the invention belongs to the technical field of bacterial detection methods, and relates to a real-time fluorescent PCR detection method and application for all 7 O antigen serotypes of Enterobacter sakazakii.
  • E. sakazakii infection can cause very serious illness, especially in premature infants. Specifically, it causes meningitis, sepsis, and fatal necrotizing enterocolitis and sepsis.
  • the Centers for Disease Control and Prevention estimates the case fatality rate for cases of sakazakii infection to be as high as 40%. Infected neonates and infants have a reported case fatality rate of 50-80%, with 20% of survivors developing severe neurological disorders. In older children, sakazakii can colonize the gastrointestinal lumen, with reported case fatality rates of 10-55%.
  • the bacteria can be isolated from many foods, including milk powder, dried vegetables, and cereal powders, but it is most commonly found in infant formula.
  • the serotype of bacteria is closely related to pathogenicity, and the pathogenicity of bacteria of different serotypes within the same species is often very different.
  • Serological typing has been widely used since the 1930s and has a history of more than 80 years. It can further classify bacteria within species/genus and is an important bacterial identification method. The most common method for identification and traceability of pathogenic bacteria.
  • traditional serological identification methods are widely used, there are still many shortcomings. Mainly include: the work of establishing a serological typing system is cumbersome and the cycle is long; the production and quality control of serum is difficult; the experimental process of serological identification takes a long time (2-6 days).
  • Bacterial surface polysaccharide antigens mainly include O polysaccharide (O antigen), common antigen (CA), exopolysaccharide antigen, spore, and capsular polysaccharide (K antigen).
  • O antigen O polysaccharide
  • CA common antigen
  • K antigen capsular polysaccharide
  • bacteria can be divided into different serotypes.
  • the O-antigen diversity of different serotypes of the same bacterium is determined by the genetic diversity of genes encoding various enzymes that synthesize O-antigen. These genes are often clustered at fixed sites on the genome and are called O-antigen genes. cluster.
  • the O antigen gene clusters of E. sakazakii are all located between the galF and gnd genes. According to the diversity of their O antigens, they can be divided into seven O antigen serotypes.
  • TaqMan-MGB real-time fluorescence PCR Real-time fluorescence PCR
  • the principle of TaqMan-MGB real-time fluorescence PCR is that after mixing the Taqman probe labeled with fluorescein with the template DNA, the thermal cycle of high temperature denaturation, low temperature renaturation, and suitable temperature extension is completed, and the polymerization is followed.
  • the Taqman probe that is complementary to the template DNA is cut off, and the fluorescein is free in the reaction system and emits fluorescence under specific light excitation.
  • the amplified target gene fragments increase exponentially, and the type and content of the detected sample can be characterized by detecting the corresponding fluorescence signal intensity that changes with the amplification in real time and obtaining the Ct value.
  • TaqMan fluorescent probe is an oligonucleotide probe, its 5' end carries a fluorophore, such as FAM, TET, VIC, HEX, etc., and the 3' end carries a quencher group, such as TAMRA, BHQ, etc.
  • a specific fluorescent probe is added at the same time as a pair of primers.
  • the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'- of Taq enzyme The 3' exonuclease activity degrades the probe and separates the reporter fluorophore and the quencher fluorophore, so that the fluorescence monitoring system can receive the fluorescent signal, that is, each time a DNA chain is amplified, a fluorescent molecule is formed. , the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.
  • TaqMan-MGB probe is a new technology improved on the basis of TaqMan probe in recent years. It adds MGB molecule at the 3′ end of the probe, which can increase the annealing temperature (Tm value) of the probe, thereby It can distinguish 1 base difference, complete pairing, and there is a fluorescent signal; as long as one base does not match, there is no signal.
  • the probes were first designed to be 15-30 nt in length with a Tm of 68-70°C.
  • the probes were synthesized by AuGCT Biotechnology Corporation (Beijing, China) with 6-carboxyfluorescein (FAM) and minor groove binding (MGB) moieties at the 5' and 3' ends, respectively.
  • the primers are about 25nt in length and have a Tm between 55-60°C.
  • the resulting PCR products were between 50-200 bp.
  • Primers were synthesized by Invitrogen (Shanghai, China). It may be better if the 3' end of the forward primer is 1-20 nt (usually 10 nt or less) from the 5' end of the probe.
  • amplification reactions were performed in a total volume of 25 ⁇ L, including 12.5 ⁇ L Premix Ex Taq TM (Takara), 0.3 ⁇ L each 10 ⁇ M forward and reverse primers, 0.3 ⁇ L 10 ⁇ M probe, 0.2 ⁇ L ROXII, 0.3 ⁇ L nucleic acid DNA of the sample to be tested and 11 ⁇ L ddH 2 O.
  • One protocol included an initial denaturation step of 3 min at 95°C, followed by 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 45 s in triplicate on a 7500 real-time PCR system (Applied Biosystems, Foster City). , CA, USA), followed by detection and result analysis using an instrument such as ABI 7500. The Ct value is less than 26 and the amplification curve appears, the sample is judged to be positive, otherwise it is negative.
  • the present invention provides a method and application for real-time fluorescent PCR detection of Enterobacter sakazakii O antigen.
  • the invention provides nucleotide molecules screened from the wzy genes of Enterobacter sakazakii O1-O2 and O4-O6 and/or the wzx genes of O3 and O7 respectively, which can be used to prepare detection marks of Enterobacter sakazakii thing.
  • the present invention also provides a primer or a combination thereof, which has the nucleotide sequence shown in any of SEQ ID No. 1-14.
  • the nucleotide sequence comprises:
  • the upstream primer for amplifying the Enterobacter sakazakii O1 type wzy gene has the nucleotide sequence shown in SEQ ID No.1; the downstream primer for amplifying the Enterobacter sakazakii O1 type wzy gene has the nucleotide sequence shown in SEQ ID No.2 the nucleotide sequence shown;
  • the upstream primer for amplifying the O2-type wzy gene of Enterobacter sakazakii has the nucleotide sequence shown in SEQ ID No.3; the downstream primer for amplifying the O2-type wzy gene of Enterobacter sakazakii has the nucleotide sequence shown in SEQ ID No.4 the nucleotide sequence shown;
  • the upstream primer for amplifying the Enterobacter sakazakii O3 type wzx gene has the nucleotide sequence shown in SEQ ID No.5;
  • the downstream primer for amplifying the Enterobacter sakazakii O3 type wzx gene has the nucleotide sequence shown in SEQ ID No.6 the nucleotide sequence shown;
  • the upstream primer for amplifying Enterobacter sakazakii O4 type wzy gene has the nucleotide sequence shown in SEQ ID No.7;
  • the downstream primer for amplifying Enterobacter sakazakii O4 type wzy gene has the nucleotide sequence shown in SEQ ID No.8 the nucleotide sequence shown;
  • the upstream primer for amplifying the Enterobacter sakazakii O5 type wzy gene has the nucleotide sequence shown in SEQ ID No.9; the downstream primer for amplifying the Enterobacter sakazakii O5 type wzy gene has the nucleotide sequence shown in SEQ ID No.10 the nucleotide sequence shown;
  • the upstream primer for amplifying Enterobacter sakazakii O6-type wzy gene has the nucleotide sequence shown in SEQ ID No.11; the downstream primer for amplifying Enterobacter sakazakii O6-type wzy gene has the nucleotide sequence shown in SEQ ID No.12 the nucleotide sequence shown;
  • the upstream primer for amplifying the wzx gene of Enterobacter sakazakii O7 has the nucleotide sequence shown in SEQ ID No.13; the downstream primer for amplifying the wzx gene of Enterobacter sakazakii O7 has the nucleotide sequence shown in SEQ ID No.14 The nucleotide sequence shown.
  • the present invention also provides probes corresponding to the primers or their combination, the positions of which are located between the corresponding upstream and downstream primers on the gene, and each probe contains a 5' reporter dye (FAM) and a 3' non-reporter dye. Fluorescence quencher (NFQ), conjugated to the minor groove binder (MGB) at the 3' end.
  • FAM 5' reporter dye
  • NFQ Fluorescence quencher
  • MGB minor groove binder
  • sequences of the probes are shown in SEQ ID Nos. 15-21.
  • the present invention also provides applications of the primers or combinations thereof and/or the probes in preparing detection reagents or detection tools for different O antigen serotypes of Enterobacter sakazakii.
  • the detection means comprise detection kits or chips.
  • the present invention also provides detection reagents or detection tools, including the primers or combinations thereof and/or the probes and detection-acceptable aids or carriers.
  • the detection means comprise detection kits.
  • the present invention also provides a method for detecting different O antigen serotypes of Enterobacter sakazakii.
  • the sample to be tested is amplified and detected by the primers or combinations thereof and/or the probes.
  • Fig. 1 The self-detection results of each system in the real-time fluorescent PCR detection system of Enterobacter sakazakii O antigen for the corresponding serotype strain;
  • the invention discloses a method and application for real-time fluorescent PCR detection of Enterobacter sakazakii O antigen, and those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the purpose of the present invention is to provide a real-time fluorescent PCR detection technology that can identify all 7 O antigen serotypes of Enterobacter sakazakii, and to provide an effective technical means for the rapid detection of this pathogenic bacteria.
  • the invention relates to a Taqman primer sequence, an MGB probe sequence and a real-time fluorescent PCR detection method which are respectively specific to different O antigen serotype Enterobacter sakazakii. in:
  • Described Taqman primer is 2 DNA fragments selected respectively in the wzy gene of Enterobacter sakazakii O1-O2 type, O4-O6, and the wzx gene of O3 type, O7 type, its sequence is such as SEQ ID NO:1- shown in SEQ ID NO:14.
  • the MGB probes were selected from the wzy genes of Enterobacter sakazakii O1-O2 and O4-O6, and the wzx genes of O3 and O7, respectively, and were screened within two Taqman primers corresponding to the corresponding serotypes. obtained, and containing a 5' reporter dye (FAM) and a 3' non-fluorescent quencher (NFQ), conjugated to a minor groove binder (MGB) at the 3' end, the sequence of which is as SEQ ID NO: 15-SEQ ID NO:21.
  • FAM 5' reporter dye
  • NFQ 3' non-fluorescent quencher conjugated to a minor groove binder
  • the invention further discloses a real-time fluorescent PCR detection system for detecting different O antigen serotype strains of Enterobacter sakazakii in a sample and its application.
  • the composition and concentration of the above system are as follows:
  • the technology provided by the present invention has good accuracy and can accurately detect all 7 O antigen serotypes of Enterobacter sakazakii.
  • CT value is less than 26
  • strains of other serotypes the CT value is greater than the standard of 26 or no
  • the amplification curve appears, which can be judged as a negative result. As shown in the table below and Figure 2.
  • the technology provided by the present invention has high sensitivity, and can accurately detect 10 pg of Enterobacter sakazakii genomic DNA.
  • the invention mainly provides a technical means for detecting all O antigen serotypes of Enterobacter sakazakii, a common pathogen in food, especially in infant formula milk powder by using molecular biological method, and the main difficulty lies in specific Taqman primers and MGB Screening and identification of probes. Its positive effects are:
  • the sources of Enterobacter sakazakii strains used in the present invention are as follows.
  • the method for real-time fluorescent PCR detection of Enterobacter sakazakii O antigen provided by the present invention and the raw materials and reagents used in the application can be purchased from the market.
  • oligosaccharide unit processing enzyme gene There are three types of genes in the O antigen synthesis gene cluster of Enterobacter sakazakii: monosaccharide synthase gene, glycosyltransferase gene and oligosaccharide unit processing enzyme gene (wzx and wzy).
  • the oligosaccharide unit processing enzyme gene has the most type specificity, followed by the glycosyltransferase gene, and the monosaccharide synthase gene is less specific. Therefore, the wzy genes of Enterobacter sakazakii types O1-O2 and O4-O6, and the wzx genes of types O3 and O7 were selected as specific genes, and specific primers and probes were designed.
  • TaqMan MGB probes and primers were designed based on the specific gene sequence of each serotype.
  • the GC% content of primers and probes is between 40-70%, avoiding the formation of loop hairpin structures, self-dimers and cross-dimers.
  • the specificity of primers and probes was verified by comparing their sequences to all sequences in GenBank using BLAST searches.
  • the probes were first designed to be 15-30 nt in length with a Tm of 68-70°C. At the 5' end of the probe, the presence of Gs that could cause fluorescence quenching is avoided.
  • the probe was synthesized by AuGCT Biotechnology Corporation (Beijing, China), with 6-carboxyfluorescein (FAM) and non-fluorescent quencher (NFQ) at the 5' and 3' ends, respectively, and a minor groove binder ( MGB) conjugated.
  • FAM 6-carboxyfluorescein
  • NFQ non-fluorescent quencher
  • the primers are about 25nt in length and have a Tm between 55-60°C.
  • the resulting PCR products were between 50-200 bp.
  • Primers were synthesized by Invitrogen (Shanghai, China). It may be better if the 3' end of the forward primer is 1-20 nt (usually 10 nt or less) from the 5' end of the probe.
  • the primer sequences are shown in SEQ ID No. 1-14.
  • the probe sequences are shown in SEQ ID Nos. 15-21.
  • Example 2 Take the nucleic acid solution extracted in Example 2 as the template of real-time fluorescent PCR reaction, add O1-O7 each real-time fluorescent PCR reaction system respectively, put into real-time fluorescence quantitative gene amplification instrument (this embodiment selects the 7500 type real-time real-time fluorescent quantitative gene amplification instrument of American ABI company. Fluorescence quantitative PCR instrument), and the reaction was carried out under the following conditions.
  • Bacterial genomic DNA extraction kit product number DP302 of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract bacterial genomic DNA according to its operation steps.

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Abstract

一种用于阪崎肠杆菌O抗原实时荧光PCR检测方法及应用。主要是采用MGB探针的实时荧光PCR反应快速检测体系对阪崎肠杆菌7个O抗原血清型进行检测。以阪崎肠杆菌O1-O2型、O4-O6的wzy基因，以及O3型、O7型的wzx基因为靶基因，设计了并筛选了针对每一个血清型的Taqman特异性引物及MGB探针，并建立了实时荧光PCR检测方法。该方法可以准确、快速、灵敏地对阪崎肠杆菌全部7个O抗原血清型菌株进行分子生物学检测。

Description

一种用于阪崎肠杆菌O抗原实时荧光PCR检测的方法及应用
本申请要求于2020年07月08日提交中国专利局、申请号为202010649411.9、发明名称为“一种用于阪崎肠杆菌O抗原实时荧光PCR检测的方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于细菌检测方法技术领域，涉及用于阪崎肠杆菌全部7个O抗原血清型的实时荧光PCR检测方法及应用。
背景技术
阪崎肠杆菌感染能引起非常严重的疾病，尤其是在早产儿中。具体为引起脑膜炎、脓毒症和致死性坏死性小肠结肠炎、败血症。疾病和预防控制中心估计阪崎氏杆菌感染病例的病死率高达40％。据报道，受感染的新生儿和婴儿的病死率为50-80％，其中20％的幸存者发展为严重的神经障碍。在年龄较大的儿童中，阪崎杆菌可以在胃肠腔定殖，报告显示由此引起的病死率为10-55％。该菌可从很多食品中分离出来，包括奶粉、干蔬菜和谷物粉，但最常见于婴儿配方奶粉。
细菌的血清型与致病性密切相关，同一种内不同血清型细菌的致病性往往存在着很大差异。血清学分型自上世纪30年代开始被广泛使用，至今已有超过80年的历史，可在种/属内将细菌进一步分类，是一种重要的细菌鉴定方法，目前，血清学鉴定方法是使用最为普遍的致病菌鉴定和溯源方法。传统血清学鉴定方法虽被广泛使用，但仍存在诸多缺陷。主要包括：建立血清学分型系统的工作繁琐、周期长；血清的生产和质控较为困难；血清学鉴定的实验过程耗时较长(2-6天)等。
细菌表面多糖抗原主要包括O多糖(O抗原)、普通抗原(CA)、胞外多糖抗原、芽孢、以及荚膜多糖(K抗原)等。基于上述表面多糖抗原的多样性，细菌可被分为不同的血清型。同一细菌不同血清型的O抗原多样性，是由于编码合成O抗原的各种酶的基因的遗传多样性决定的， 这些基因往往成簇地位于基因组上的固定位点，被称作O抗原基因簇。阪崎肠杆菌的O抗原基因簇均位于galF和gnd基因之间，根据其O抗原的多样性，可以分为7个O抗原血清型。
TaqMan-MGB实时荧光PCR(Real-time fluorescence PCR)技术原理是，将标记有荧光素的Taqman探针与模板DNA混合后，完成高温变性，低温复性，适温延伸的热循环，并遵守聚合酶链反应规律，与模板DNA互补配对的Taqman探针被切断，荧光素游离于反应体系中，在特定光激发下发出荧光。随着循环次数的增加，被扩增的目的基因片段呈指数规律增长，通过实时检测与之对应的随扩增而变化荧光信号强度，求得Ct值，即可表征检测样品的类型和含量。
TaqMan荧光探针是一种寡核苷酸探针，其5'末端携带荧光基团，如FAM、TET、VIC、HEX等，3'端携带淬灭基团，如TAMRA、BHQ等。PCR扩增时在加入一对引物的同时加入一个特异性的荧光探针，探针完整时，报告基团发射的荧光信号被淬灭基团吸收；PCR扩增时，Taq酶的5'－3'外切酶活性将探针酶切降解，使报告荧光基团和淬灭荧光基团分离，从而荧光监测系统可接收到荧光信号，即每扩增一条DNA链，就有一个荧光分子形成，实现了荧光信号的累积与PCR产物形成完全同步。
TaqMan-MGB探针是近年来在TaqMan探针的基础上改进的一种新技术，它在探针3′端增加了MGB分子，这种物质能够提高探针的退火温度(Tm值)，从而使它能够分辨1个碱基的差别，完全配对，有荧光信号；只要有一个碱基不匹配，就没有信号。探针的设计首先为15-30nt长度，Tm为68-70℃。探针由AuGCT Biotechnology Corporation(中国北京)合成，分别在5'和3'末端具有6-羧基荧光素(FAM)和小沟结合(MGB)部分。引物的长度约为25nt，Tm在55-60℃之间。产生的PCR产物在50-200bp之间。引物由Invitrogen(中国上海)合成。如果正向引物的3'末端距离探针的5'末端1-20nt(通常为10nt或更少)，则可能更好。
利用Taqman MGB PCR检测时，扩增反应在25μL的总体积中进行，包括12.5μL Premix Ex Taq TM(Takara)，0.3μL各10μM正向和反向引物， 0.3μL 10μM探针，0.2μL ROXII，0.3μL待检测样品的核酸DNA和11μL ddH 2O。一个方案包括在95℃下进行3分钟的初始变性步骤，然后在95℃下变性15秒，在60℃下退火45秒进行40个循环，一式三份进行7500实时PCR系统(Applied Biosystems，Foster City，CA，USA)，随后利用仪器(如ABI 7500)进行检测和结果分析。Ct值<26且出现扩增曲线，则判断样品为阳性，否则为阴性。
发明内容
有鉴于此，本发明提供了一种用于阪崎肠杆菌O抗原实时荧光PCR检测的方法及应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了从阪崎肠杆菌O1-O2型、O4-O6的wzy基因和/或O3型、O7型的wzx基因中分别筛选得到的核苷酸分子，可用于制备阪崎肠杆菌检测标志物。
基于上述，本发明还提供了引物或其组合，具有如SEQ ID No.1～14任意所示的核苷酸序列。
在本发明的一些具体实施方案中，所述核苷酸序列包括：
扩增阪崎肠杆菌O1型wzy基因的上游引物，具有如SEQ ID No.1所示的核苷酸序列；扩增阪崎肠杆菌O1型wzy基因的下游引物，具有如SEQ IDNo.2所示的核苷酸序列；
扩增阪崎肠杆菌O2型wzy基因的上游引物，具有如SEQ ID No.3所示的核苷酸序列；扩增阪崎肠杆菌O2型wzy基因的下游引物，具有如SEQ IDNo.4所示的核苷酸序列；
扩增阪崎肠杆菌O3型wzx基因的上游引物，具有如SEQ ID No.5所示的核苷酸序列；扩增阪崎肠杆菌O3型wzx基因的下游引物，具有如SEQ IDNo.6所示的核苷酸序列；
扩增阪崎肠杆菌O4型wzy基因的上游引物，具有如SEQ ID No.7所示的核苷酸序列；扩增阪崎肠杆菌O4型wzy基因的下游引物，具有如SEQ IDNo.8所示的核苷酸序列；
扩增阪崎肠杆菌O5型wzy基因的上游引物，具有如SEQ ID No.9所示的核苷酸序列；扩增阪崎肠杆菌O5型wzy基因的下游引物，具有如SEQ IDNo.10所示的核苷酸序列；
扩增阪崎肠杆菌O6型wzy基因的上游引物，具有如SEQ ID No.11所示的核苷酸序列；扩增阪崎肠杆菌O6型wzy基因的下游引物，具有如SEQ IDNo.12所示的核苷酸序列；
扩增阪崎肠杆菌O7型wzx基因的上游引物，具有如SEQ ID No.13所示的核苷酸序列；扩增阪崎肠杆菌O7型wzx基因的下游引物，具有如SEQ IDNo.14所示的核苷酸序列。
本发明还提供了所述的引物或其组合对应的探针，其在基因上的位置均位于对应的上、下游引物之间，每条探针含有5'报告染料(FAM)和3'非荧光猝灭剂(NFQ)，在3'末端与小沟结合物(MGB)缀合。
在本发明的一些具体实施方案中，所述探针的序列如SEQ ID No.15～21所示。
本发明还提供了所述的引物或其组合和/或所述的探针在制备用于不同O抗原血清型阪崎肠杆菌的检测试剂或检测工具中的应用。
在本发明的一些具体实施方案中，所述检测工具包括检测试剂盒或芯片。
更重要的是，本发明还提供了检测试剂或检测工具，包括所述的引物或其组合和/或所述的探针以及检测可接受的助剂或载体。
在本发明的一些具体实施方案中，所述检测工具包括检测试剂盒。
在本发明的一些具体实施方案中，以体积份计：包括
Figure PCTCN2020135549-appb-000001
Figure PCTCN2020135549-appb-000002
Figure PCTCN2020135549-appb-000003
本发明还提供了不同O抗原血清型阪崎肠杆菌的检测方法，取待测样本经所述的引物或其组合和/或所述的探针扩增，检测。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1阪崎肠杆菌O抗原实时荧光PCR检测体系中每个体系对于相应血清型菌株的自身检测结果；
图2阪崎肠杆菌O抗原实时荧光PCR检测体系中每个体系的特异性实验检测结果。
具体实施方式
本发明公开了一种用于阪崎肠杆菌O抗原实时荧光PCR检测的方法及应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对 本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明的目的在于提供一种可对阪崎肠杆菌全部7个O抗原血清型进行鉴定的实时荧光PCR检测技术，为这种致病菌的快速检测提供有效的技术手段。
本发明涉及一种对不同O抗原血清型阪崎肠杆菌分别特异的Taqman引物序列，MGB探针序列及实时荧光PCR检测方法。其中：
所述的Taqman引物是在阪崎肠杆菌O1-O2型、O4-O6的wzy基因，以及O3型、O7型的wzx基因中分别选取的2个DNA片段，其序列如SEQ ID NO:1-SEQ ID NO:14所示。
Figure PCTCN2020135549-appb-000004
Figure PCTCN2020135549-appb-000005
所述的MGB探针是从阪崎肠杆菌O1-O2型、O4-O6的wzy基因，以及O3型、O7型的wzx基因中分别选取，且在相应血清型对应的2条Taqman引物内部筛选得到的，且含有5'报告染料(FAM)和3'非荧光猝灭剂(NFQ)，在3'末端与小沟结合物(MGB)缀合，其序列如SEQ ID NO:15-SEQ ID NO:21所示。
Figure PCTCN2020135549-appb-000006
Figure PCTCN2020135549-appb-000007
本发明进一步公开了用于检测样品中阪崎肠杆菌不同O抗原血清型菌株的实时荧光PCR检测体系及其应用。上述体系的组成成分和浓度如下：
Figure PCTCN2020135549-appb-000008
Figure PCTCN2020135549-appb-000009
实验结果显示：
(1)本发明提供的技术准确性好，可以实现对阪崎肠杆菌全部7个O抗原血清型的准确检测。对于阪崎肠杆菌O1型，其对应的O1体系实时荧光PCR检测结果Ct＝20.25；对于阪崎肠杆菌O2型，其对应的O2体系实时荧光PCR检测结果Ct＝18.92；对于阪崎肠杆菌O3型，其对应的O3体系实时荧光PCR检测结果Ct＝18.97；对于阪崎肠杆菌O4型，其对应的O4体系实时荧光PCR检测结果Ct＝16.88；对于阪崎肠杆菌O5型，其对应的O5体系实时荧光PCR检测结果Ct＝18.96；对于阪崎肠杆菌O6型，其对应的O6体系实时荧光PCR检测结果Ct＝14.13；对于阪崎肠杆菌O7型，其对应的O7体系实时荧光PCR检测结果Ct＝16.64。可见，每个体系对于各自对应血清型的实时荧光PCR检测CT值均小于26的标准，可判断为阳性结果。如图1所示。
(2)本发明提供的技术特异性好，O1-O7每个体系除了可以检测自身对应的血清型外(CT值小于26)，对于其他血清型的菌株，CT值均大于26的标准或无扩增曲线出现，可判断为阴性结果。如下表和图2所示。
表1阪崎肠杆菌实时荧光PCR特异性反应结果
Figure PCTCN2020135549-appb-000010
Figure PCTCN2020135549-appb-000011
(3)本发明提供的技术灵敏度高，可以实现对10pg阪崎肠杆菌基因组DNA的准确检测。
本发明主要提供了利用分子生物方法，对食品中，特别是婴儿配方奶粉中的常见致病菌-阪崎肠杆菌全部O抗原血清型进行检测的技术手段，主要难点在于特异性Taqman引物和MGB探针的筛选与确定。其积极效果在于：
(1)首次公开了利用分子生物学手段，对阪崎肠杆菌全部O抗原血清型进行检测的技术手段，对于该菌的临床检测和流行病学监控提供了有效方法。
(2)检测灵敏度高：对于基因组DNA的检测灵敏度可达10pg。
(3)检测时间短：利用该技术手段，自获得基因组DNA或纯培养菌落计，可在3个小时内完成检测。
本发明所用到的阪崎肠杆菌菌种来源如下。
表2本发明涉及的细菌菌种
血清型 实验室编号
O1 G2539
O2 G2356
O3 G2726
O4 G2594
O5 G2704
O6 G2706
O7 G2592
本发明提供的一种用于阪崎肠杆菌O抗原实时荧光PCR检测的方法及应用中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1 引物、探针的设计
1、特异基因的筛选
阪崎肠杆菌的O抗原合成基因簇中，存在3类基因，分别是：单糖合成酶基因、糖基转移酶基因和寡糖单位处理酶基因(wzx和wzy)。其中，寡糖单位处理酶基因最具有型别的特异性，糖基转移酶基因次之，单糖合成酶基因特异性较差。因此，选择阪崎肠杆菌O1-O2型、O4-O6的wzy基因，以及O3型、O7型的wzx基因作为特异基因，进行特异引物和探针的设计。
2、引物和探针的设计
使用Primer Express 3.0，根据每个血清型的特异基因序列设计TaqMan MGB探针和引物。引物和探针的GC％含量在40-70％之间，避免了环发夹结构，自身二聚体和交叉二聚体的形成。通过使用BLAST搜索将它们的序列与GenBank中的所有序列进行比较来验证引物和探针的特异性。
探针的设计首先为15-30nt长度，Tm为68-70℃。在探针的5'末端，避免了可能引起荧光猝灭的G的存在。探针由AuGCT Biotechnology Corporation(中国北京)合成，分别在5'和3'末端具有6-羧基荧光素(FAM)和非荧光猝灭剂(NFQ)，且在3'末端与小沟结合物(MGB)缀合。
引物的长度约为25nt，Tm在55-60℃之间。产生的PCR产物在50-200bp之间。引物由Invitrogen(中国上海)合成。如果正向引物的3'末端距离探针的5'末端1-20nt(通常为10nt或更少)，则可能更好。
引物序列如SEQ ID No.1～14所示。
探针序列如SEQ ID No.15～21所示。
实施例2 样本核酸的提取
1、针对获得的纯培养细菌，采用以下方式进行处理：
(1)挑取细菌单菌落至10μL去离子水中，或过夜培养的细菌菌液10μL，沸水浴中处理15min。
(2)置于冰上1min后，8000rpm离心1min。
(3)取3μL上清液作为下一步实时荧光PCR反应的模板。
2、取婴儿配方奶粉10g，采用以下方式进行处理：
(1)将样品置于20mLLB培养基中，37度震荡培养3h。
(2)取1mL(1)中的培养物，8000rpm离心5min，弃去上清。
(3)加入500μL去离子水，重悬混匀，8000rpm离心5min，弃去上清。
(4)加入100μL去离子水，沸水浴中处理15min。
(5)置于冰上1min后，8000rpm离心1min。
(6)取3μL上清液作为下一步实时荧光PCR反应的模板。
实施例3 实时荧光PCR扩增检测
以实施例2提取的核酸溶液作为实时荧光PCR反应的模板，分别加入O1-O7每个实时荧光PCR反应体系，放入实时荧光定量基因扩增仪(本实施例选择美国ABI公司的7500型实时荧光定量PCR仪)，按如下条件进行反应。
反应条件：
Figure PCTCN2020135549-appb-000012
检测结果中，某个体系的CT值小于26，则判断样品中含有该体系对应血清型的阪崎肠杆菌。
实施例4 灵敏度检测
(1)将每个血清型的阪崎肠杆菌菌株接种至5mL LB培养基中，37℃，180转/分钟的摇床中过夜培养，收集2mL菌体。
(2)利用天根生化科技(北京)有限公司的细菌基因组DNA提取试剂盒(产品号DP302)，按照其操作步骤进行细菌基因组DNA的提取。
(3)用NanoDrop OD仪对提取的基因组浓度进行测定，并将其浓度调整为3300ng/μL，并依次逐级梯度稀释。如此，加入对应反应体系的基因组DNA分别为100ng、10ng、1ng、100pg、10pg和1pg。
(4)按照实施例3的反应步骤进行实时荧光PCR反应，结果表明：对于基因组DNA的检测灵敏度为10pg。
以上对本发明所提供的一种用于阪崎肠杆菌O抗原实时荧光PCR检测的方法及应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (9)

  1. 引物或其组合，其特征在于，具有如SEQ ID No.1～14任意所示的核苷酸序列。
  2. 如权利要求1所述的引物或其组合，其特征在于，所述核苷酸序列包括：
    扩增阪崎肠杆菌O1型wzy基因的上游引物，具有如SEQ ID No.1所示的核苷酸序列；扩增阪崎肠杆菌O1型wzy基因的下游引物，具有如SEQ ID No.2所示的核苷酸序列；
    扩增阪崎肠杆菌O2型wzy基因的上游引物，具有如SEQ ID No.3所示的核苷酸序列；扩增阪崎肠杆菌O2型wzy基因的下游引物，具有如SEQ ID No.4所示的核苷酸序列；
    扩增阪崎肠杆菌O3型wzx基因的上游引物，具有如SEQ ID No.5所示的核苷酸序列；扩增阪崎肠杆菌O3型wzx基因的下游引物，具有如SEQ ID No.6所示的核苷酸序列；
    扩增阪崎肠杆菌O4型wzy基因的上游引物，具有如SEQ ID No.7所示的核苷酸序列；扩增阪崎肠杆菌O4型wzy基因的下游引物，具有如SEQ ID No.8所示的核苷酸序列；
    扩增阪崎肠杆菌O5型wzy基因的上游引物，具有如SEQ ID No.9所示的核苷酸序列；扩增阪崎肠杆菌O5型wzy基因的下游引物，具有如SEQ ID No.10所示的核苷酸序列；
    扩增阪崎肠杆菌O6型wzy基因的上游引物，具有如SEQ ID No.11所示的核苷酸序列；扩增阪崎肠杆菌O6型wzy基因的下游引物，具有如SEQ ID No.12所示的核苷酸序列；
    扩增阪崎肠杆菌O7型wzx基因的上游引物，具有如SEQ ID No.13所示的核苷酸序列；扩增阪崎肠杆菌O7型wzx基因的下游引物，具有如SEQ ID No.14所示的核苷酸序列。
  3. 如权利要求1或2所述的引物或其组合对应的探针，其特征在于，其在基因上的位置均位于对应的上、下游引物之间，每条探针含有5'报告 染料(FAM)和3'非荧光猝灭剂(NFQ)，在3'末端与小沟结合物(MGB)缀合；
    所述探针的序列如SEQ ID No.15～21所示。
  4. 如权利要求1或2所述的引物或其组合和/或如权利要求4所述的探针在制备用于不同O抗原血清型阪崎肠杆菌的检测试剂或检测工具中的应用。
  5. 如权利要求4所述的应用，其特征在于，所述检测工具包括检测试剂盒。
  6. 检测试剂或检测工具，其特征在于，包括如权利要求1或2所述的引物或其组合和/或如权利要求3所述的探针以及检测可接受的助剂或载体。
  7. 如权利要求6所述的检测试剂或检测工具，其特征在于，所述检测工具包括检测试剂盒。
  8. 如权利要求6或7所述的检测试剂或检测工具，其特征在于，以体积份计：包括
    Figure PCTCN2020135549-appb-100001
    Figure PCTCN2020135549-appb-100002
    Figure PCTCN2020135549-appb-100003
  9. 不同O抗原血清型阪崎肠杆菌的检测方法，其特征在于，取待测样本经如权利要求1或2所述的引物或其组合和/或如权利要求3所述的探针扩增，检测。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005078068A1 (en) * 2004-02-12 2005-08-25 The University Of Queensland Live attenuated salmonella for use as vaccine
CN102154270A (zh) * 2011-01-11 2011-08-17 天津生物芯片技术有限责任公司 一种对阪崎肠杆菌o抗原特异的核苷酸及其应用
WO2019016188A1 (en) * 2017-07-19 2019-01-24 Glaxosmithkline Biologicals Sa IMMUNOGENIC COMPOSITION
CN111676304A (zh) * 2020-07-08 2020-09-18 南开大学 一种用于阪崎肠杆菌o抗原实时荧光pcr检测的方法及应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005078068A1 (en) * 2004-02-12 2005-08-25 The University Of Queensland Live attenuated salmonella for use as vaccine
CN102154270A (zh) * 2011-01-11 2011-08-17 天津生物芯片技术有限责任公司 一种对阪崎肠杆菌o抗原特异的核苷酸及其应用
WO2019016188A1 (en) * 2017-07-19 2019-01-24 Glaxosmithkline Biologicals Sa IMMUNOGENIC COMPOSITION
CN111676304A (zh) * 2020-07-08 2020-09-18 南开大学 一种用于阪崎肠杆菌o抗原实时荧光pcr检测的方法及应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LU YAN,, WANG MIAO,FANG YU-GUO,WANG MIN,WANG YU,ZHAO CHANG-LI: "Quantitative PCR Method for Detecting Enterobacter sakazakii in Infant Powder", CHINA DAIRY INDUSTRY, vol. 36, no. 7, 25 July 2008 (2008-07-25), XP055886510 *
SIL EUN KANG, YONG SUK NAM, KWANG WON HONG: "Rapid Detection of Enterobacter Sakazakii with TaqMan-MGB Probe Based on Real-Time PCR Assay", JOURNAL OF ENVIRONMENTAL & OCCUPATIONAL MEDICINE, vol. 17, no. 3, 1 January 2007 (2007-01-01), pages 516 - 519, XP055886504 *
WANG MIN, ARBATSKY NIKOLAY P., XU LINGLING, SHASHKOV ALEXANDER S., WANG LEI, KNIREL YURIY A.: "O antigen of Franconibacter pulverisG3872 (O1) is a 4-deoxy-d-arabino-hexose-containing polysaccharide synthesized by the ABC-transporter-dependent pathway", MICROBIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, vol. 162, no. 7, 1 July 2016 (2016-07-01), Reading , pages 1103 - 1113, XP055886493, ISSN: 1350-0872, DOI: 10.1099/mic.0.000307 *

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